Device and process for producing fiber products and fiber products produced thereby

ABSTRACT

The present invention is directed to a fiber, preferably bone fiber, having a textured surface, which acts as an effective binding substrate for bone-forming cells and for the induction or promotion of new bone growth by bone-forming cells, which bind to the fiber. Methods of using the bone fibers to induce or promote new bone growth and bone material compositions comprising the bone fibers are also described. The invention further relates to a substrate cutter device and cutter, which are effective in producing substrate fibers, such as bone fibers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.15/494,001, filed Apr. 21, 2017 which is a Continuation of U.S. patentapplication Ser. No. 12/692,879, filed on Jan. 25, 2010; which is aDivisional Application of U.S. patent application Ser. No. 10/606,208,filed on Jun. 26, 2003, now U.S. Pat. No. 7,744,597; which claimspriority to and the benefit of U.S. Provisional Patent Application Ser.No. 60/391,323, filed Jun. 26, 2002, which are all hereby incorporatedherein by reference in their entireties.

FIELD OF THE INVENTION

This invention relates to a cutting device for cutting a substrate,processes for the production of substrate fibers, and the substratefibers produced. Suitable substrates include but are not limited to bonetissue, including allogenic and xenogenic cortical bone. The fibers arecut from a substrate using the device, such that an individual fiberproduced has a length that is typically greater than 10 to 200 times itswidth and thickness. The invention further relates to compositionsincluding bone fibers and other agents, including, for example,bioactive agents, including stent cells, which bind to the bone fibersand are induced to form new bone.

BACKGROUND OF THE INVENTION

Ground demineralized cortical and cancellous bone have been widely usedin the induction of new bone formation for the treatment of a variety ofclinical pathologies. Typically, the bone materials are obtained fromhuman or animal sources, ground and demineralized. Such bone has beendemonstrated over the past two decades to induce new bone formation whenimplanted in animal models, to stimulate elevated levels of the enzymealkaline phosphatase, and to contain extractable amounts of bioactivemolecules, such as bone morphogenetic proteins (BMPs).

The ground demineralized bone matrix (DBM) has also been calleddemineralized bone (DMB), and demineralized freeze-dried bone allograft(DFDBA). DFDBA materials are provided for clinical use in a freeze-driedstate. DBM (or DMB) can be provided for clinical use in either afreeze-dried state or as a hydrated state—usually in some form of anaqueous carrier, for example, glycerol in GRAFTON™ (GRAFTON™ is aregistered trademark of Osteotech, Inc., Shrewsbury, N.J.), pluronicpolymer in DYNAGRAFT™ (DYNAGRAFT™ is a registered trademark of GenSciRegeneration Technologies, Inc., Irvine, Calif.), and collagen inOPTIFORM™ (OPTIFORM™ is a registered trademark of RegenerationTechnologies, Inc., Alachua, Fla.). These various commercially availabledemineralized bone products primarily contain demineralized corticalpound bone distributed for clinical applications. The use of carrierswith demineralized bone particles are more acceptable to cliniciansbecause such particles acquire a static charge in the dry state makingthem difficult to dispense into containers and following rehydration,the clinician typically has difficulties in getting the bone particlesto remain at the implant site and in a compacted state wherein they arepresumed to be most osteoinductive. DBM is considered to beosteoinductive if it induces the formation of new bone, for example, atthe site of clinical application. By adding carriers to the DBM, thebiomaterials become easier to aliquot into containers and tend to remaintightly aggregated at the implant site making them easier to handle.

The osteoinductive nature of DBM arises from the interaction betweenbone-forming cells and the DBM. Such interaction takes place at both amolecular and physical level. At the molecular level, attachment of thebone-forming cells to the DBM involves the presence of “receptors” onthe surface of the plasma membrane of mammalian cells that bind to“ligands” present on the surface of the biomaterial. An example of thistype of attachment or binding is illustrated in the role ofRGD-containing amino acid sequences in the attachment of mammalian cellsto a wide variety of molecules present within matrices of tissues. TheRGD amino acid sequence refers to the amino acids arginine (R), glycine(G), and aspartic acid (D). Holland, et al. (Biomaterials. 1996.17(22):2147-56) described the research on a synthetic peptide,gly-arg-gly-asp-ser-pro-lys (GRGDSPK) (which includes the cell-adhesiveregion of fibronectin, and arg-gly-asp (RGD) peptide sequence covalentlybound to a dialdehyde starch (DAS) coating on a polymer surface. Theauthors concluded that the GRGDSPK/DAS-coated surface could besubstituted for an adhesive-protein coated surface in the culture ofanchorage-dependent cells.

On the other hand, binding at the physical level in the context ofsurface patterning has been described, for example, in Goodman, et al,(Biomaterials, 1996. 17(21):2087-95). Goodman et al. described clinicaland experimental investigations on manufactured surface topographiesthat have significant effects on cell adhesion and tissue integrationstating that micro- and nano-scale mechanical stresses generated bycell-matrix adhesion have significant effects on cellular phenotypicbehavior. Details of surface patterning effects on cell attachment andproliferation were described by Schmidt and Recum (Biomaterials. 1992.13(15): 1059-69) measuring macrophage responses to microtexturedsilicone. Schmidt and Recum measured the effects of seven differentsilicone surface textures on macrophage spreading and metabolic activityin vitro. Variables of the textured arrays important, to cell spreadingand metabolic activity included size, spacing between, depth, density,and orientation of the individual surface events and the roughness ofthe surfaces. It was found that pattern dimensions of about 5 micronstextures were associated with small cells, whereas a smooth (untextured)surface was associated with large cells. The authors put forth ahypothesis that included a possible mechanism of how a micrometer-sizedsurface texture could modify cell function.

There are thus several issues pertinent to the ability of implanted bonecompositions to induce the formation of bone. These issues includeproviding an environment suitable for the infiltration of cells, aconfined environment that restricts the diffusion of synthesizedmatrix-forming molecules (for example, collagens, proteoglycans, andhyaluronins), promotes cell attachment to DMBs, and includes thepresence of bioactive molecules (for example BMPs). Additionally, themethod for making bone fibers for these bone implanted compositions inan efficient and consistent manner is addressed by the presentinvention.

SUMMARY OF THE INVENTION

The present invention is directed to a fiber, preferably bone fiber,having a textured surface, which acts as an effective binding substratefor bone-forming cells and for the induction or promotion of new bonegrowth by bone-forming cells, which bind to the fiber. The bone fiber ofthe present invention may be demineralized or mineralized, or may beused in a composition comprising a combination of demineralized andmineralized bone fibers and bone particles. The bone fibers of theinvention may be made from any type of bone, such as allogenic orxenogenic bone. Preferably, the bone fiber is made from cortical bone orcancellous bone, more preferably cortical bone. The bone fiber may be ofany length. Preferably, the bone fiber has an average length of fromabout 1 mm to about 100 mm, an average width of from about 0.5 mm toabout 2.5 mm, and an average thickness of from about 0.2 mm to about 1.4mm. The fiber may then be processed according to known processes. In apreferred embodiment, the bone is freeze-dried.

The present invention further is directed to bone material compositionscomprising the bone fibers of the present invention. In a preferredembodiment of this aspect of the invention, the bone materialcomposition comprises a bone fiber and bone-forming cells, wherein thebone fiber has a textured surface, which acts as an effective bindingsubstrate for bone-forming cells, and wherein the composition induces orpromotes new bone formation from the bone-forming cells bound to thebone fiber. Preferably, the bone-forming cells are selected from stemcells, connective tissue progenitor cells, fibroblast cells, periostealcells, chondrocytes, osteocytes, pre-osteoblasts, and osteoblasts. Mostpreferably, the bone-forming cells are stem cells. The bone fibers usedin the bone material composition may be any type of bone, includingallogenic or xenogenic bone. Preferably, the bone fibers are comprisedof cortical or cancellous bone, more preferably comprised of corticalbone. In addition, the composition may further comprise cancellous bone.The composition may further comprise both demineralized andnon-demineralized bone fibers or bone particles. The bone materialcomposition may further comprise an agent effective to initiate orpromote the induction of bone growth.

Yet another aspect of the invention is a method for inducing orpromoting bone growth. This method comprises providing a bone fiberaccording to the present invention, contacting the bone fiber tobone-forming cells, which adhere to the textured surface of the bonefiber, and wherein the binding induces or promotes new bone growth fromthe bone-forming cells. The method may further comprise contacting thebone fibers and bone-forming cells with an agent effective to initiateor promote the induction of the new bone growth. Suitable agents toinduce or promote bone growth include bone morphogenic proteins,angiogenic factors, growth and differentiation factors, mitogenicfactors, and osteogenic/chondrogenic factors. Preferably, the bone fiberused in the method is demineralized. Preferred bone-forming cellsinclude stem cells, connective tissue progenitor cells, fibroblastcells, periosteal cells, chondrocytes, osteocytes, pre-osteoblasts, andosteoblasts. Preferably, the bone-forming cells are stem cells.Moreover, the bone-forming cells may be contacted to the bone fibers viaa biological fluid. Preferably biological fluids include plasma, bonemarrow, blood, or blood products.

According to another aspect of the present invention a cutter isprovided for producing substrate fibers. The cutter preferably includesa leading edge designed to make initial contact with the substrate and atrailing edge. The trailing edge preferably is configured such that itis raised above the leading edge by a prescribed height. The cutterincludes a cutting surface upon which a blade section is disposed. Theblade section is used to cut the substrate. At least one substratechannel may be provided near the blade section in order to direct thesubstrate fibers away from the substrate.

According to one exemplary embodiment of the present invention, theblade section can include at least one row of teeth designedspecifically for cutting the substrate. Furthermore, each tooth can beconfigured with at least one predetermined, cutting angle to reducestress and achieve desired substrate properties. For example, onespecific implementation of the invention provides a preferred primarycutting angle ranging from 3-6. Preferably the primary cutting angle canbe selected to be approximately 4. A secondary cutting angle can also beprovided. The secondary cutting angle can vary between 10-18, but ispreferably selected to be approximately 14.

According to another aspect of the invention, a substrate cutting deviceis provided. The substrate cutting device includes a base and a tower.The base further includes a cutter that can be moved along apredetermined cutting path. A substrate chute extends through the basein order to position the substrate in a location where it will be incontact with the cutter. The tower includes a lower surface, whichcontains a recess. The recess can be aligned with the substrate chute. Aclamping mechanism is provided to keep the substrate in contact with thecutter during the cutting process. The substrate cutting device canfurther include a fiber receptacle to receive the substrate fibers afterthey have been cut.

According to one exemplary embodiment of the present invention, the baseis mounted on a slide mechanism, which moves along the predeterminedcutting path. An actuation unit, such as a pneumatic actuator, can beused to supply the force necessary for moving the slide mechanism.According to one specific implementation of the present invention, thefirst actuation generates a force ranging between 600 lbs-900 lbs, andpreferably about 750 lbs. A second actuation unit can also be providedto control the clamping mechanism. The second actuation unit can beconfigured to generate a force ranging from 150 lbs-250 lbs, andpreferably about 200 lbs. The present invention can also include acomputer controller for controlling operation of the substrate cuttingdevice, including the first and second actuation units. For example, thecomputer controller can be used to adjust the force applied by the firstactuation unit and/or adjust the speed at which the slide mechanism ismoving. The computer controller can also be used to adjust the forceapplied on the substrate during the cutting process.

According to another aspect of the present invention, a method forcutting a substrate comprises the steps: placing the substrate into asubstrate cutting device; applying a predetermined force on thesubstrate; moving a substrate cutter along a grain direction of thesubstrate; cutting substrate fibers from the substrate; detecting whenthe substrate has reached a predetermined minimum thickness; andterminating the process if the substrate has reached the predeterminedminimum thickness.

The present invention is further directed to the substrate fibersproduced using the substrate cutting device of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates fiber bone having a ribbon-like structure producedusing an apparatus of the present invention.

FIGS. 2A, 2B and 2C illustrate scanning electron photomicrographs ofrehydrated bone fibers at a magnification of 268×. (FIG. 2A), 37×. (FIG.2B) and 13×. (FIG. 2C), which depict the parallel striations along thegrain of the bone fibers and the serrated edges and grooves which arebelieved to foster attachment sites for bone-forming cells.

FIG. 3 depicts a histology slide stained with Hematoxylin and Eosin ofbone fibers of the present invention when implanted intramuscularly inan athymic (nude) mouse bioassay. The arrows (.fwdarw.) indicate sitesof new bone formation.

FIG. 4 photographically illustrates the bone fibers when combined withcancellous particle bone to create a composite matrix as used to bindstem cells from blood, bone marrow, or similar cellular composition.

FIG. 5 is a side elevational view of a substrate cutting deviceaccording to an exemplary embodiment of the present invention.

FIG. 6 is a top plan view of the substrate cutting device.

FIG. 7 illustrates an operations cart that can be used to support thesubstrate cutting device and store various components.

FIG. 8 illustrates a top portion of the base of the substrate cuttingdevice.

FIG. 9 is a top plan view of a cutter in accordance with an exemplaryembodiment of the present invention.

FIG. 10A is a side elevational view of the cutter.

FIG. 10B illustrates an exemplary configuration for the cutter teeth.

FIG. 11 is a perspective view of the base of the substrate cuttingdevice.

FIG. 12 is a top perspective view of a bottom portion of the base.

FIG. 13 illustrates the base of the substrate cutting de vice with allaccess doors in place.

FIG. 14 illustrates an exemplary substrate.

FIG. 15 is a perspective view of an exemplary tower for use with thesubstrate cutting device.

FIG. 16 illustrates substrate fibers produced according to oneembodiment of the present invention.

FIG. 17 illustrates alternative cutters that can be used in differentembodiments of the present invention.

FIG. 18 illustrates an exemplary configuration for use with one of thealternative cutters shown in FIG. 17.

FIG. 19 is a flow chart showing the steps performed when cuttingsubstrates.

FIGS. 20A and 20B are histology slides stained with H&E of human bonefibers of the present invention (FIG. 20A) and human particle bone usedas a control (FIG. 20B) implanted intramuscularly in an athymic (nude)mouse bioassay as set forth in Example 3 and, after 28 days, explantedand fixed in buffered formalin. The arrows (.fwdarw.) indicate sites ofnew bone formation.

DETAILED DESCRIPTION

The invention provides a bone fiber having surface properties that offera suitable environment for the attachment of infiltrating cells, suchthat they can attach (normal mammalian cells are “attachment dependent”meaning they do not typically proliferate or maintain syntheticfunctions unless attached to a solid matrix) and synthesize bonematrix-forming molecules. Appropriate attachment surfaces can alsocontribute to the stimulation of cells to proliferate, differentiate,and to synthesize appropriate bone matrix-forming molecules.

The present invention is also directed to a method of making the bonefibers of the present invention involving the use of an apparatussuitable for cutting bone to produce fibers having the enhancedcell-binding surface to increase the bone-forming induction propertiesof demineralized bone material and to facilitate formation of a matrixsuitable for perfusion, percolation, and infusion of viscous cellmaterials into the matrix.

Finally, the present invention is directed to an apparatus suitable forcutting a substrate. The apparatus includes a unique arrangement thatallows the substrate to be cut into fibers having consistent propertiesfor a particular application. A special cutter is used to cut the fibersalong a grain direction of the substrate in order to produce substratefibers. The apparatus includes various safety features, such as sensorsto detect whether all access doors are shut prior to commencingoperation. If a sensor is triggered during operation, the apparatus isimmediately powered down in order to prevent an operator from beingharmed. The present apparatus can also include a computer controller tocontrol various operations.

I. Definitions

The terms used herein are given their plain, ordinary meaning asunderstood by those having ordinary skill in the art, unless otherwisedefined herein.

The “bioactive agents” of the present invention refer to the agentscapable of initiating and inducing the differentiation and/orproliferation of bone cells and/or the induction of bone cell growth.The bioactive agents may include, for example, bone morphogenicproteins, stem cells, blood, blood elements, bone marrow and bone marrowextracts, platelets and platelet extracts, homogenates of skin and skinhomogenate extracts, growth factors, selenium and transferrin, calciumsalts, and CYMETRA™ (CYMETRA™ is a registered trademark of LifeCellInc., New Jersey).

“Bone formation” as used in the present invention, refers to the act ofthe bone-forming cells taking the form of bone cells, bone, cartilage,osteoids, and bone matrices.

The term, “bone material composition,” means a composition comprisingthe bone fibers or bone fibers plus anorganic or inorganic componentsmixed with the bone fibers of the present invention and bone-formingcells. Typically, this combination has physical characteristics thatallow infusion of visous materials such as bone marrow andosteoinductive effect so as to allow the bone-forming cells to form intonew bone cells under appropriate conditions.

The “cutting cycle” is a single forward plus backward stroke of thecutter across the substrate as disclosed herein.

A “cutting event” is the complete cutting run of a load chute of asubstrate.

“Demineralization” refers to the act of removing minerals from tissuescontaining minerals. The demineralization may be conducted by processesknown in the art.

The “fiber bone” or “bone fiber” of the present invention is the fibermade from bone by shaving or cutting along the length of the bone toprovide the bone fiber its textured surface to which bone-forming cellsmay bind and the induction of bone growth may be initiated underappropriate conditions.

“Osteoinductive” shall mean the ability to induce or promote theformation of new bone either in vivo or in vitro. For example, the bonefibers of the present invention have been found to induce or promote theformation of new bone by bone-forming cells attached to its surface. Theinduction of new bone may be fostered by the presence of bioactiveagents that assist in the initiation of this induction process.

The “substrate” of the present invention may be any material, i.e.,non-biological or biological materials, which may be cut using thecutting device of the present invention. Where the substrate is bone,for example, the bone fibers act as a material upon which an organismsuch as bone-forming cells may grow or attach.

II. Bone Fibers and Methods of Inducing Bone Formation

The bone fibers of the present invention have the ability to induce orpromote bone formation and have properties-particularly suitable as acomponent in bone implants. The bone fibers can be made from cortical,or cancellous bone, and torn any source, i.e., allograft or xenograft,by the essentially linear cutting from a bone-cutting device. Theessentially linear cuttings, i.e., cuttings along the grain direction ofthe bone, result in bone fibers that optionally curl with the cuttingprocess to form ribbon-like structures such as shown in FIG. 1. Thefibers of the present invention preferably have a textured surface, asshown in FIGS. 2A, 2B, and 2C, having serrated edges and grooves as wellas parallel striations, which provide an improved binding substrate towhich bone-forming cells may attach. It is believed that this texturedsurface provides more available attachment sites to which bone-formingcells may adhere. Upon attachment, these cells can differentiate to formnew bone and proliferate as new bone cells. Thus, the fibers of thepresent invention enhance the ability of bone-forming cells to bind tothem so as to enhance the formation of new bone.

Fibers can be cut from any substrate that is capable of being cut usingthe device. Suitable substrates include non-biological materials, andbiological materials. For example, suitable substrates include bone,bone tissue, plasticized bone, plasticized soft tissue, freeze-driedbone, freeze-dried soft tissue, frozen bone, frozen soft tissue, newlyformed bone, implant, bone, and associated cells, bone marrow, bonemarrow-like tissue, cartilage, and cartilage-like tissues. Preferably,the substrate is bone tissue. Any type of bone may be used, such asallogenic and xenogenic bone. The bone tissue may be derived from anymammalian source, but is preferably human.

Production of bone fibers begins with the procurement of bone suitableto the preparation of fiber bone and includes any bone in an animal,such as bone diaphyseal shafts of long bones, for example the femur,tibia, humerus, ribs, radius, fibula. In humans, such bones are composedprimarily of cortical bone tissue, but may also include cancellous bone.

The bone used to make the bone fibers may be processed in known mannersprior to forming the fibers of the present invention. For example, thebone may be treated with enzymes to partially digest the organiccomponents of the bone, such as collagenase, papain, protease,hyaluronidase, endonuclease, lipase, and/or phosphatase, or organicacids, such as acetic or citric acid. Alternatively, the bone may bepartially digested by breaking or fragmenting the covalent bonds in theindividual collagen molecules contained in the demineralized bone. Oncethe bone is cleaned of associated soft tissue, it can then be optionallycut into lengths and shapes appropriate for use in the cutting device.Alternatively the fiber bone can be cut directly from the shaft portionswhere the cutting blade can be attached to a manual (hand-held) cuttingblade holder.

The bone tissue is used to form the bone fibers by contacting the bonetissue with an instrument capable of cutting along the length or alongthe grain direction of the bone tissue. The cutting instrument should becapable of cutting to provide serrated edges and grooves on theresulting bone fibers, which act as a surface-enhanced binding substratefor bone-forming cells. It has been found that bone-forming cells havean increased ability to attach to these bone fibers. While not intendingto be bound by particular theory, it is believed that the edges andgrooves formed on the bone fibers of the present invention provide moreattachment sites to which the bone-forming cells may bind.

Cell binding to the fiber bone may be easily observed and quantitatedusing any number of assay methods known in the art. For example, cellpopulations present in any number of suspension formats, for example,bone marrow, concentrated platelets, blood, liver homogenates, etc., canbe incubated with the fiber bone. The fiber bone can then be separatedfrom the cell solution(s), gently washed to remove loosely adherentcells and other biomaterials present in the cell suspensions. The cellsretained on the fiber bone can be quantitated using the traditionalmethyltetrazolium (MTT) assay where an insoluble chromogenic compound isformed due to the presence of metabolically viable cells (activemitochondrial enzymes) where fiber bone incubated with the suspensionformat lacking cells is used as a control. Alternatively, the fiber bonecan be fixed with any number of fixatives, for example, formalin, andthe DNA in adherent cells stained for visualization using lightmicroscopy. The phenotypic identity, for example, fibroblasts,chondroblasts, osteoblasts, etc. can be verified using traditionalenzyme assays such as alkaline phosphatase activity stains. Fibroblasts(less differentiated cells) stain only minimally for this enzyme,whereas chondrogenic and osteogenic cells stain heavily for this enzyme.

In the method of inducing new bone formation, the bone fibers of thepresent invention may be used in either a mineralized or demineralizedstate or a combination thereof. Whether mineralized or demineralized,the bone fibers have the textured surface to which the bone-formingcells may efficiently attach. In the case of demineralized bone fibers,the ribbon-like structures typically unwind into essentially linearstrips of bone.

The bone fibers may be of any length, width, and thickness as deemednecessary or useful for its intended use. For example, the fibers may bethe length of bone tissue from which they are being made. Alternatively,the fibers may be designed to be cut at shorter lengths to accommodatetheir use in particular bone implants. Bone fibers preferably haveaverage length of from about 1 mm to about 100 mm, an average width offrom about 0.5 mm to about 2.5 mm, and an average thickness of fromabout 0.2 mm to about 1.4 mm, more preferably having an average lengthof from about 20 mm to about 30 mm, an average width of from about 1.0mm to about 2.0 mm, and an average thickness of from about 0.4 mm toabout 0.8 mm. Furthermore, it is noted that the length of the fibersproduced according to the invention may be substantially greater thanthe width and thickness of the fibers. For example, the bone fibers mayhave a length that is greater than about 10 to about 200 times its widthand thickness, preferably about 40 to about 100 times its width andthickness. As will be described further herein, the cutting apparatus ofthe present invention may be modified to accommodate any desired length,width or thickness of the fibers.

For demineralization, the mineral content of the bone fibers may beremoved using any known process for demineralization causing the bonefibers to be demineralized. Preferably, the bone fibers aredemineralized to contain calcium at a level of from about 0.5 wt % toabout 4.5 wt %, more preferably from about 1.0 wt % to about 4.0 wt %,and most preferably from about 1.5 wt % to about 3.5 wt %, for example,as disclosed in U.S. Pat. Nos. 6,189,537 and 6,305,379; and co-pendingU.S. patent application Ser. Nos. 09/655,711 and 10/180,989, thedisclosures of which are herein incorporated by reference in theirentireties. Once demineralized, the bone fibers may optionally becombined with agents including for example, biological carriers,bioactive agents, or other agents including for example, surface activeagents, preservatives including for example glycerol, and inorganicmineral compositions, either before or after further processing, suchfurther processing including but not limited to, freeze-drying, terminalsterilization processes, and/or retaining as a hydrated fiber bone inthe presence or absence of preserving agents, or combined immediatelyprior to implantation in a patient. Moreover, the bone fibers of thepresent invention may be further combined with other carriers and agentsas one having ordinary skill in the art would appreciate for the useDMBs. For example, suitable biological, carriers include collagen,gelatin, saccharides, fibrin, fibrinogen, alginates, hyaluronins,methylcelluloses, and biologically compatible thixotropic agents.Suitable bioactive agents include but are not limited to, bonemorphogenic proteins, stem cells, blood, blood elements, bone marrow andbone marrow extracts, platelets and platelet extracts, homogenates ofskin and skin homogenate extracts, growth factors, selenium andtransferrin, calcium salts, and CYMETRA™.

Production of demineralized bone biomaterials and the induction of newbone by these biomaterials are described in U.S. Pat. Nos. 5,275,054,6,189,537 and 6,305,379, of which are herein incorporated by referencein their entireties. The bone fibers of the present invention may induceor promote new bone formation by serving as a source of one or morechemoattractants that diffuse from the bone biomaterials to cause cellsto migrate to the implanted bone fibers wherein cells adhere to the boneparticles (normal mammalian cells are “attachment dependent,” meaningthey typically require attachment to some surface in order to functionmetabolically) and differentiate towards a chondrocyte (cartilageforming) or osteocytic (bone forming) phenotype. In accordance with thepresent invention, it is believed that surface characteristics of thebone fibers of the present invention render the fibers more accessibleand are a more accepting substrate to receive and bind bone-formingcells. Thus, the surface characteristics of the bone fibers may resultin improved cell attachments, and consequently, act as a means forselectively attaching cartilage or bone-forming cells from a mixedpopulation of cells, such as are present in platelet-rich plasma, blood,blood products, or bone marrow.

In accordance with the present invention, the surface patterning presenton the bone fibers preferably contains parallel striations, cracks, andseriated edges and grooves to which cells may attach. This surfacepattern of the bone fibers of the present invention permits a multitudeof cells to bind to the bone fibers allowing less specific cells to bindand grow based on the functional properties of the fibers.

In a preferred embodiment of the present invention, the surfacepatterning of the bone fibers is created by the bone-cutting device ofthe invention as described herein. As illustrated in FIGS. 2A, 2B, and2C, the cutting “bits” (blades) of the fiber bone-cutting device cause amicro-fractured surface with specific patterns of parallel surfacestriations on the cut surface of the fiber bone. Thus, in addition tothe normal osteoinductive properties of demineralized bone (as describedin U.S. Pat. No. 6,189,537), the bone fibers of the present invention,whether demineralized or not demineralized, present a micro-patternedsurface that is not only biocompatible with bone-forming cells, but alsopresents a surface conducive to cellular attachment, cell spreading, andcell proliferation/differentiation (or maintenance of phenotype of analready differentiated cell). The available surface area of fiber boneproduced by the present fiber bone cutting device, as compared to normalparticle bone (produced by impact fragmentation), is greater, isbiocompatible, and presents the surface patterning conducive to cellularattachment, proliferation, and differentiation. Due to the individualand multiple cutting bits present on a cutter present within the fiberbone cutting device, the multiplicity of patterns on the multiple fiberbone fibers produced would contribute to maximal availability of optimalsurface patterning for cellular attachment Thus, the fibers of thepresent invention can have variable sizes, spacing between striations,depths, density, and orientations. Preferably, the fibers are cut alongthe grain to obtain a greater durability of the fiber.

The “bone-forming cells” or “bone-matrix forming cells” of the presentinvention are those cells suitable for the induction of new boneformation when infiltrated with the bone fibers of the present inventionand include those cell types suitable for differentiating into bonecells or suitable for forming a matrix similar to osteoid of forming newbone. Suitable cell types may include differentiated, partiallydifferentiated, or undifferentiated cells. For example, cell typesinclude, but are not limited to stem cells, connective tissue progenitorcells, fibroblast cells, periosteal cells, chondrocytes, osteocytes,pre-osteoblasts, and osteoblasts. Preferably, the stem cells aremultipotent, the fibroblast, cells are undifferentiated, the periostealcells are partially differentiated, and the chondrocytes or osteocytesare differentiated.

Preferably, the bone-forming cells are stem cells. Stem cells representa population of cells present throughout the body of mammals that areundifferentiated possessing the potential for differentiating intovirtually any other, more differentiated, cell in the body. For thisreason, stem cells represent a unique opportunity to repair and/orremodel damaged tissues such as broken bones, abraded cartilage, skin,etc.

Moreover, the bone-forming cells may be tissue progenitor cells. Forexample, U.S. Pat. Nos. 5,824,084 and 6,049,026 (and U.S. patentapplication 2002/0161449) describe kits and composite bone graftscontained in the kits, wherein the composite bone graft(s) are designedto contain an enriched population of connective tissue progenitor cellsand a greater number of connective tissue progenitor cells per unitvolume than found in the original bone marrow aspirate.

In one aspect of the invention, the bone fibers of the present inventionallow for the formulation of “bone material compositions” comprising thebone fibers for use in bone implants. These bone material compositionsprovide increased accessibility of the bone fibers to bone-forming cellsby permitting suitable voids through which viscous solutions of plateletrich plasma, bone marrow, blood or blood products may flow. For example,the bone fibers may be demineralized and compacted to form a bonematerial composition suitable for implantation. Because the bone fibersof the present invention are easily handled without breaking apart, thebone fibers may be molded to create an implantable composition, whichretains its shape in the implant and further has appropriate spacingthrough which such solutions comprising bone-forming cells may pass.These bone material compositions may further have integrated thereinother components, such as inorganic particles, organic particles, ormore specifically non-demineralized cancellous or cortical bone chunks,which may increase the ability of such solutions to flow through thecomposition by providing structural spacing of the fiber bone. Undersuch conditions, the surface of the fiber bone fibers would be presentedto the infiltrating bone marrow/platelet rich plasma preparations topromote cellular attachment, selectively concentrating the cells mostappropriate to the formation of bone or cartilage when the bone materialcomposition is then implanted into some clinical site in the body. Suchex-vivo exposure of the bone fiber biomaterials to osteogenic orchondrogenic cells would serve to concentrate cells that would normallybe expected to migrate into the implanted materials through the normalchemoattractive properties of demineralized bone. Thus, thispre-implantation exposure of cells to the bone fibers should reduce thetime required for the initiation of new bone formation and lessen theclinical times needed to affect a repair of the damaged site in thebody, i.e. a broken bone or fusion site in an intervertebral fusionprocedure for repair of cervical or lumbar complications in the spine.Other suitable components for integration into the bone materialinclude, but are not be limited to, inorganics such as particulatecalcium salts, such as calcium phosphates, calcium sulfates, and/orcalcium carbonates, organics such as particulate skin, particulatecartilage, particulate tendons and ligaments, particulate dextrans,particulate alginates, and particulate resorbable and non-resorbablesynthetic polymeric materials.

The bone material compositions may be formed in manners known in theart. In one embodiment of the present invention, the bone fibers of thepresent invention and bone-forming cells are preferably placed in abioreactor capable of simulating the nutrient flow and waste removalpresent within an implant site. The flow of nutrient solutions into,through, and out of the bioreactor permit the associated grounddemineralized bone and bone-forming cells to form into bone or bone-likebiomaterial suitable for transplantation. In the present instance, thebioreactor use aspect of the present invention would simulate theactions of the fibers and fiber bone compositions when used clinically.The process of making bone in a bioreactor is described in ApplicationNo. 60/466,772, for example, which is herein incorporated by reference.

In another aspect of the invention, the bone fibers of the presentinvention have exhibited superior properties for the formation of boneimplants. Bone implants may be formed using the bone fibers of thepresent invention based on their ability to be easily handled formolding, retaining its shape, and allowing appropriate spacing forbiological solutions to pass therethrough even upon compaction. Forexample, the fibers may be hydrated, which renders then pliable andmalleable, but capable of retaining its shape without losing durability.In fact, the fibers have been shown to retain its integrity even uponhydration, molding, and subjection to other bone implant-formingtreatments. Therefore, the bone fibers of the present invention havesuperior properties making them ideal for the formation of boneimplants.

In another aspect of the invention, the bone fibers can be used alone orin conjunction with a bone material composition and placed in a suitablecontainer through which blood, blood products, bone marrow, or plateletrich plasma can be induced to flow through such that the cells capableof adhering to the bone material composition, specifically the fiberbone, are suitably concentrated for implantation into a site in the bodywherein the formation of new bone is desired.

III. Production of Bone Fibers

Referring to FIG. 5, a device for cutting substrates in accordance withthe present invention will now be described. The substrate cuttingdevice 100 includes a base 110, a tower 112, and a cutter 114. Withcontinued reference to FIG. 5 and additional reference to FIG. 6, thebase 110 of the substrate cutting device 100 includes a slide mechanism116 which travels along a predetermined cutting path. Preferably, thecutting path is along with, or substantially parallel to, a grain 164(see FIG. 14) of the substrate being cut. A pair of guide rods 118 isused to control the direction of the slide mechanism 116 duringoperation. A plurality of bearings 120 are also used to slidably engagethe guide rods 118.

A first actuation unit 122 generates to force necessary to move theslide mechanism 116. According to the disclosed embodiment of theinvention, the first actuation unit 122 is pneumatically operated. Itshould be noted, however, that the first actuation unit 122 can also beoperated hydraulically, electrically, and or mechanically depending onthe specific requirements. As illustrated in FIGS. 5 and 6, the firstactuation unit 122 includes an air cylinder 124 that receivespressurized air to generate the forces necessary for moving the slidemechanism 116. Referring additionally to FIG. 7, a plurality ofpneumatic cables 126 are used to supply air to the air cylinder 124.Preferably, the air is pressurized at an external location andtransferred to the substrate cutting device 100. According to such anarrangement, the pressurized air can optionally be processed in order tomaintain sterile environment, when necessary. FIG. 7 also illustrates afoot pedal 128 which can be used to control the operation of thesubstrate cutting device 100. A computer controller 188 can also beprovided to monitor and control operation of the substrate cuttingdevice 100.

According to the disclosed embodiment of the invention, the firstactuation unit 122 is configured to generate a force ranging from 600lbs to 900 lbs. Preferably, the first actuation unit 122 generate aforce ranging from 700 lbs to 800 lbs. Most preferably, the force isapproximately 750 lbs. Additionally, the force can be varied duringoperation of the substrate cutting device 100, or it can be maintainedat a constant level. For example, according to one embodiment of theinvention, the computer controller 188 can be used vary the forceapplied by the first actuation unit 122 by reducing the amount of forceapplied during a return stroke and increasing the force applied during acutting stroke.

As best illustrated in FIG. 8, the top surface of the base 110 includesa cutter access 130 which allows an operator to mount the cutter 114within the substrate cutting device 100. The top surface of thesubstrate cutting device 100 also includes a substrate chute 152designed to appropriately position a substrate 362 (see also FIG. 14) sothat it may be engaged by the cutter 116. The dimensions of thesubstrate chute 152 can vary depending on the specific substrate andproduct desired. The various parts of the substrate cutting device 100can be secured using a variety of means such as, for example, threadedfasteners 150 or any appropriate method capable of providing thestrength and/or function necessary for proper operation. FIG. 8 alsoillustrates that the cutter 114 is rotated such that it is offset fromthe cutting path when mounted on the slide mechanism 116. The specificrotational offset can be selected based on a variety of factorsincluding, but not limited to, the type of substrate, the specificarrangement of the blade sections on the cutter, and the amount of forcebeing applied by the first actuation unit.

Turning to FIG. 9, the details of the cutter 114 will now be described.The cutter 114 includes a leading edge 134 and a trailing edge 136.During a cutting stroke, the leading edge 134 is the first portion ofthe cutter 114 to reach the substrate 162. It should be noted, however,that the leading edge 134 will not necessarily contact the substrate162. The cutter 114 includes a plurality of blade sections 138 disposedon its surface. Each blade section 138 contains two rows of teeth 140.Depending on the specific application, desired product, and substrate,the cutter 114 can include a single blade section 138 or multiple bladesections 138 (as shown in FIG. 9). Additionally, a single row of teeth,or multiple rows of teeth may be provided.

According to the disclosed embodiment of the present invention, when thecutter 114 is mounted on the slide mechanism 116, the cutter surface issubstantially flush with the surface of the slide mechanism 116. Such aconfiguration advanfageouslv minimizes movement of the substrate 162during operation. Furthermore, as shown in FIG. 10A, the trailing edge136 of the cutter 114 is raised by a predetermined amount. Preferably,this predetermined amount is approximately equal to the height of theteeth 140 in the blade section 138 in order to further minimize possiblemovement of the substrate 162 during operation. Referring additionallyto FIG. 13, once the cutter 114 has been securely mounted to the slidemechanism 116, a cutter access door 132 is used to prevent access to thecutter 114 during operation of substrate cutting device 100.

Referring to FIGS. 10A and 10B, each tooth 140 in the blade sections 138can include one or more cutting angles. In addition, the cutting anglecan be Independently selected for each individual tooth 140. Moreparticularly, one tooth may include a single cutting angle while anadjacent tooth can include two cutting angles, and yet another adjacenttooth can contain three cutting angles. As disclosed in FIG. 10B, eachtooth 140 contains a primary cutting angle 142 and a secondary cuttingangle 144. The primary cutting angle 142 can be selected to be in therange of 3 to 6. Preferably, the primary cutting angle 142 is selectedto be approximately 4.

The secondary cutting angle 144 can be selected in the range of 10 to18. The secondary cutting angle 144 can also range from 12 to 16.Preferably, however, the secondary cutting angle 144 is selected to beapproximately 14. FIGS. 10A and 10B also illustrate a cutting height 146for the teeth 140. The cutting height 146 can vary depending on thespecific operation and/or product desired. For example, the cuttingheight 146 can be used to define the thickness of fibers produced. Thecutter 114 also includes a plurality of fiber channels 148 to allowpassage of substrate fibers after being cut. The fiber channels 148 canbe generally selected to correspond with the number of blade sections138. More particularly, the cutter 114 is designed such that the cutsubstrate fibers pass directly through the fiber channel 148.Furthermore, the fiber channel 148 can be sized to assist in theproduction of substrate fibers having required features for a particularproduct. For example, by selecting an appropriate width for the fiberchannel 148, the cut fibers can be prevented from curling back into thefiber channel 148 and possibly breaking prematurely. Likewise, selectionof an appropriate depth for the fiber channel 148 can prevent fibersfrom curling into adjacent fiber channels 148.

Turning now to FIGS. 11 and 12, additional features of the base 110 willbe discussed. The substrate cutting device 100 can include a liberreceptacle 154 for collecting substrate fibers that have been cut. FIG.16 illustrates a plurality of fibers that have been collected in thefiber receptacle 154. The fiber receptacle 154 is inserted into the base110 such that it is aligned with the cutter 114 and the fiber channels148. Accordingly, the cut fibers will fall directly into the fiberreceptacle 154. A plurality of guides 156 (best seen in FIG. 12) areprovided to properly align the fiber receptacle 154. A locking clip 158can optionally be used to secure the fiber receptacle 154 in place. Itshould be noted, however, that various other methods and arrangementscan be used to secure the fiber receptacle 154 in place. A receptacledoor 160 is used cover the fiber receptacle 154 and prevent accessduring operation of the substrate cutting device 100. The receptacledoor also includes a reflector (not shown), such as the reflector 196 onthe slide mechanism 116, that allows sensor device 190 d to determinewhether the receptacle door 160 is closed.

Turning again to FIG. 6, the base 110 includes a plurality of sensordevices 190(a-d). The sensor devices 190 are preferably optical, but canincorporate various other detection methods as is well known. Sensordevice 190 a detects when the slide mechanism 116 has reached the rest(or home) position. Sensor device 190 b detects when the slide mechanism116 has completed the cutting stroke. Sensor device 190 c detects thepresence of the cutter access door 132. As previously indicated, sensordevice 190 d detects the presence of the receptacle door 160. Undernormal circumstances, if sensor devices 190 c and 190 d return a fault,then operation of the substrate cutting device 100 is immediatelyhalted. Additionally, sensor devices 190 a and 190 b can be used tomonitor movement of the slide mechanism 116.

Referring to FIG. 15, with additional reference to FIG. 5, the tower 112includes a lower surface 166 having a recess 168 therethrough. Therecess 168 is positioned such that if can be aligned with the substratechute 152. The tower 112 includes an opening 170 on a front portionthereof. The opening 170 is used to allow placement of the substrate 162within the substrate chute 152. Once the substrate 162 is in placed, inthe substrate chute 152, a clamping mechanism 178 is used to keep thesubstrate 162 in contact with the cutter 114.

A second actuation unit 172 is used to generate the force necessary tooperate the second actuation unit 172. As illustrated in the embodimentof the invention shown in FIG. 5, the second actuation unit 172 ispneumatically controlled. It should be noted however, that hydraulic,mechanical, electrical, and other control systems can be used, so longas they are capable of supplying the force necessary to operate theclamping mechanism 178. According to the disclosed embodiment of theinvention, the second actuation unit 172 is capable of generating aforce ranging from 150 lbs to 250 lbs. Preferably, the second actuationunit 172 generates a force of approximately 200 lbs. Similar to thefirst actuation device 122, the force can be varied during operation ofthe substrate cutting device 100 or it can be maintained at a constantlevel. Additionally, the computer controller 188 can be used to monitorand/or vary the force applied by the second actuation unit 172.

As shown in FIG. 5, the clamping mechanism 178 includes a contactsurface 180 that engages the substrate 162. According to a preferredembodiment of the invention, the contact surface 180 contains aplurality of grooves 182 designed to increase the friction force betweenthe clamping mechanism 178 and substrate. The tower also includes a door184 which prevents access during operation. One or more locating pins186 can be used to quickly and easily align the tower 112 with the base110. Additionally, a clamp stopper 192 can be used to prevent theclamping mechanism 178 from traveling too far and corning into contactwith the cutter 114.

According to the disclosed embodiment of the invention, the tower 112includes three sensor devices 190(e-g). Sensor device 190 e detects whenthe clamping mechanism 178 is in the “up” (or home) position. Sensordevice 190 f detects when the clamping mechanism 178 is in the vicinityof the clamp stopper 192. Accordingly, sensor device 190 f and the clampstopper 192 both function to prevent accidental contact with the cutter114. Sensor device 190 g detects the presence of the door 184. If anerror signal is obtained from sensor device 190 g, then operation of thesubstrate cutting device 100 is immediately halted.

FIGS. 17 and 18 illustrates a plurality of wheel type cutters 198 (orwheel cutters) that can be used with an alternative embodiment of thepresent invention. The wheel cutters 198 are mounted on a base such thatthey may be rotated and brought into contact with the substrate. Thewheel cutters 198 can be designed with various features to producefibers having desired properties. For example, the thread depth of thewheel cutters 198 can be increased in order to produce fibers having anincreased thickness. Varying the pitch of the wheel cutter 198 willeffect the length and curvature of the fibers produced. As shown in FIG.19, a substrate path 200 is used to bring the substrate in contact withthe wheel cutter 198. When the pitch of the wheel cutter 198 rotatesclockwise relative to the substrate, a “pulling” effect results. Thisrequires less force on the substrate during the cutting process, andproduces fibers that are short and curly. When the pitch of the wheelcutter 198 rotates counter-clockwise relative to the substrate, agreater force must be applied in order to maintain contact with thewheel cutter 198. However, the resulting fibers can be longer and willhave very consistent dimensions.

FIG. 19 is a flowchart illustrating the steps performed to producefibers in accordance with an exemplary embodiment of the presentinvention. The process begins at step S300. At step S310, the substrateis loaded into the substrate chute. At step S312, all of the accessdoors (i.e., cutter access door, receptacle door, and tower door) areclosed. At step S314, the sensor devices are checked to verify that allaccess doors are currently closed. If any of the access doors are open,control passes to step S316. The system waits a predetermined amount oftime, for example 10 seconds, and checks the sensor devices again.Alternatively, the system could continuously check the sensor devicesuntil all access doors are closed.

Once all access doors are determined to be closed, control passes tostep S318. The clamp is then activated. As previously discussed, thiscan be accomplished by second actuation unit applying pressure on thesubstrate. At step S320, the cutter is activated. At step S322, thesensor devices are checked to see if the substrate size has been reducedto a thickness, which is less than a minimum value. If the substratethickness is greater than the minimum value, then control returns tostep S322 and the cuter remains active, i.e., continues to cut thesubstrate. If the substrate thickness is less than or equal to theminimum value, then the system is stopped as step S326.

As illustrated by the dashed lines, the system continuously monitors thestate of the sensor devices throughout the process. Thus, if any of theaccess doors are opened during operation of the substrate cuttingdevice, control will pass to step S316 and the system will beimmediately halted. As previously discussed, this is done, in part, toprevent injury to an operator. The system continues to operate until theeither the substrate thickness reaches the minimum size, or one of theaccess doors is opened.

EXAMPLE 1

Diaphysyl shafts (total of approximate 520 grams wet weight of bonematerial) from the long bones and ribs of a given donor (human donorinformation is confidential) were mechanically debrided (as disclosed inco-pending U.S. patent application Ser. No. 10/108,104, incorporated byreference herein) to remove associated periosteal tissue and bone marrowin the intramedulary canal. The shafts and ribs were then cut intolinear pieces with widths, thickness, and lengths approximating <45mm×<45 mm×<6 cm using a bone saw. A cut piece of cortical bone (wetweight 48 grams) was then loaded individually into the load chute of thecutting device and the clamping cylinder was locked into the closedposition. The cutting slide having the cutting blade disposed thereinwas activated and cut fiber bone was collected into the receiving bin. Atotal of 42 grams of fiber bone were accumulated during the 60 cuttingcycles (cutting cycle equals one back/forth pass of the cutler/cutterslide across the bone surface) for approximately 70 seconds withadditional bone materials being added to the feeder chute at eachcutting event. After each cutting event, another cortical shaft and/orcortical pieces were added and another cutting event was initiated. Theamount of the bone materials loaded into the chute for each cuttingevent varied. However, the number of cutting events performed weresufficient to accumulate a bulk fiber mass of approximately 490 grams(wet weight).

The cut fiber bone was stored in a sterile container in the freezer(minus 80 C.) for three days. Prior to demineralization, the cut fiberbone was cleaned with LifeNet's patented ALLOWASH™ technology. Fordemineralization, a total of 463 grams of bone materials were added tothe Pulsatile Acid Demineralization (PAD) chamber (as disclosed inco-pending U.S. patent application Ser. No. 09/655,371 hereinincorporated by reference) and demineralized to 2.5% residual calciumusing 2 cycles of 0.5 N HCl and acid volumes of 4.0 liters/cycle and 3.0liters/cycle, 1 cycle of ultrapure water of 3.0 liters/cycle, and 2cycles ultrapure water plus buffer of 3.0 liters/cycle to terminate thedemineralization process. The bone fibers were finally washed in 3.0liters of ultrapure water and stored frozen at minus 80 C in a sterilecontainer.

Aliquots of the demineralized fiber bone were removed from a sterilecontainer and transferred to the animal implantation laboratory.Aliquots of fiber bone (20 and 40 mg wet weight) were manually compactedand implanted intramuscularly into the hindquarters of athymic (nude)mice as compressed fiber bone materials using established InstitutionalAnimal Care and Use Committee approved protocols (Old DominionUniversity). After 28 days of implantation, the implanted materials wereexplained and the explants fixed in formaldehyde. The fixed explantswere embedded in paraffin and sectioned for use in preparation ofhistology slides. The prepared histology slides were stained usingHematoxylin and Eosin (H&E staining) and viewed under the microscope forinduced new bone formation. The induced new bone formation isillustrated in FIG. 3. Induced new bone formation was determined usinghistomorphometry and the bone materials were determined to have inducedsignificantly more new bone than non-osteoinductive controls, i.e. thefiber bone was deemed to be osteoinductive using the nude mouse bioassaymodel.

EXAMPLE 2 In Vitro Attachment of Fibroblast Cells to Fiber Bone

The attachment of fibroblast cells to fiber bone may be quantitatedusing the methyltetrazolium dye assay method (MIT) where metabolicactivity reduces the methyltetrazolium dye to an insoluble (chromogenic)substrate that can be quantitated using the spectrophotometer. In thisparticular assessment, cell attachment is compared with cell attachmentto particle bone (cortical bone ground, using impact fragmentation)ground to a particle size range of 250 to 710 microns, demineralized andused in equal gram equivalents.

Fibroblast cells (NIH 3T3) were chosen for the study in that these cellsrepresent relatively undifferentiated cells present in the body and arepresumed to represent those cells that primarily migrate to the site ofimplantation of demineralized bone such as used in nude (athymic) mouseimplant studies to assess the osteoinductivity of demineralized bone.

Fibroblast cells (1-5×10⁵ cells/ml) grown in RPMI 1640 tissue culturemedium (supplemented with 10%, by volume, fetal calf serum (FCS) andglutamine) were harvested from the T-75 culture flasks usingtrypsinization. The residual trypsin associated with the cells put intosuspension was neutralized by resuspending the cells in fresh RPMI 1640tissue culture medium (supplemented with 10% FCS). Demineralized fiberbone (100 mg, wet weight) was aliquoted into replicate (20) 15 mlsterile centrifuge tubes and demineralized particle bone (100 mg, wetweight) was aliquoted into replicate (20) 15 ml sterile centrifugetubes. The twenty tubes of fiber bone and 20 tubes of particle bone weredivided into two groups each of 10 replicates such that one group of 10would be incubated with tissue culture medium without cells and theremaining group of 10 would be incubated with tissue culture medium withcells. Each tube received 5 mls of medium (medium containing or notcontaining cells) such that tubes receiving medium with cells receivedapproximately 5×10⁵ to 1×10⁶ cells/100 mg of demineralized bone (fiberor particle). The tubes were statically incubated at 37 C for one (1)hour, at which time the medium was decanted off of the bone and freshmedium (5 ml) added and decanted to affect a “washing” of thedemineralized bone. This “washing” process was repeated a total of threetimes. All steps were conducted using aseptic techniques such that thedemineralized bone could be incubated overnight at 37 C to permit theattached cells to proliferate.

Following the overnight incubation, the demineralized bone/medium“cells” if added in the centrifuge tube) were vigorously vortexed todislodge cells and the medium decanted to a fresh centrifuge tube. Thedislodged cells were concentrated by low speed (1,500 to 2,000 rpm in aclinical table top centrifuge) centrifugation and the medium decanted.The cell pellets were assayed using the standard MTT assay and thenumbers of cells “quantitated” by comparison to a standard curve whereknown numbers of cells were aliquoted into centrifuge tubes, centrifugedto concentration and assayed.

Background absorbance values were obtained using the demineralized bone(fiber and particle) incubated in the absence of cells. On average, thefiber bone presented 1-5×10³ cells/100 mg of bone whereas the particlebone presented approximately 2-4×10² cells/100 mg of bone, i.e., anapproximate 10-fold greater numbers of cells per unit wet weight offiber bone to particle bone.

EXAMPLE 3 In Vivo Attachment of Cells to Fiber Bone

Implantation of biomaterials into muscle pouches of athymic (nude) mice(two implants/mouse, implanted bilaterally in the gluteal region of themouse) represents the current “gold-standard” method of assessing theosteoinductivity of such biomaterials. Between 10 and 20 mg (dry weight)of biomaterials (demineralized bone in this example) are rehydrated withisotonic saline and implanted just under the fascia using a dentalamalgum tool (such as typically used by a dentist to add the filingmaterials to a cavity formed in teeth).

In this study, human “shaved” (fiber) bone and human “DMB PositiveControl” (particle) bone were implanted into muscle pouches of athymicmice (two implants/mouse and three mice per implant group). Theimplanted materials were explanted after 28 days, and the explants(explanted as “hard” nodules) were fixed in buffered formalin. Thesamples were decalcified and embedded in paraffin prior to preparationof histological sections for staining (hematoxalin/eosin; H&E). Asillustrated in FIGS. 20A (bone fibers) and 20B (control), bothdemineralized bone materials were osteoinductive, in that new boneformation was clearly visible in the histology sections. However, cellsare more clearly visible along the edges of the fiber bone materials asshown in FIG. 20A. as compared to the comparable edges of the particlebone materials as shown in FIG. 20B suggesting that cells migrating tothe implant sites were more likely to bind tightly to the fiber bonethan to the particle bone (although both demineralized bone biomaterialsinduced cells infiltrating the implant materials to differentiate into“osteoblast” or “osteoblast-like” cells and synthesize new bone matrixthat stained comparably to implant bone).

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general theprinciples of the invention and including such departures from thepresent disclosure as within known or customary practice within the artto which the invention pertains and as may be applied to the essentialfeatures hereinbefore set forth as follows in the scope of the appendedclaims. Any references including patents and published patentapplications cited herein are incorporated herein in their entirety.

1. A curled bone fiber having serrated edges and grooves.
 2. The bonefiber of claim 1, comprising demineralized bone.
 3. The bone fiber ofclaim 1, comprising non-demineralized bone,
 4. The bone fiber of claim1, comprising allogenic or xenogenic bone.
 5. The bone fiber of claim 1,comprising cortical bone.
 6. The bone fiber of claim 1, having anaverage length of from about 1.0 mm to about 100 mm.
 7. The bone fiberof claim 6, having an average length of front about 20 mm to about 30mm.
 8. The bone fiber of claim 1, having an average width of from about0.5 mm to about 2.5 mm.
 9. The bone fiber of claim 8, having an averagewidth of from about 1.0 mm to about 2.0 mm.
 10. The bone fiber of claim1, having an average thickness of from about 0.2 mm to about 1.4 mm. 11.The bone fiber of claim 10, having an average thickness is from about0.4 mm to about 0.8 mm.
 12. The bone fiber of claim 1, wherein the fiberis frozen.
 13. The bone fiber of claim 1, having a ribbon-likestructure.
 14. A bone material composition, comprising a curled bonefiber having serrated edges and grooves and one or more bone-formingcells.
 15. The bone material composition of claim 14, wherein thebone-forming cells are selected from the group consisting of stem cells,connective tissue progenitor cells, fibroblast cells, periosteal cells,chondrocytes, osteocytes, pre-osteoblasts, and osteoblasts.
 16. The bonematerial composition of claim 15, wherein the bone-forming cells arestem cells.
 17. The bone material composition of claim 14, wherein thebone fiber comprises allogenic or xenogenic bone.
 18. The bone materialcomposition of claim 14, further comprising cancellous bone.
 19. Thebone material composition of claim 14, further comprising cortical bone.20. The bone material composition of claim 14, further comprisingparticulate calcium salts selected from the group consisting of calciumphosphate, calcium sulfate, and calcium carbonate.
 21. The bone materialcomposition of claim 14, wherein the bone fiber comprises bothdemineralized and non-demineralized bone fibers.
 22. The bone materialcomposition of claim 14, wherein the composition is osteoinductive. 23.The bone material composition of claim 14, wherein the composition isfrozen.
 24. The bone material composition of claim 14, furthercomprising an agent effective to initiate the induction of bone growth.25. The bone material composition of claim 14, having a ribbon-likestructure.
 26. The bone material composition of claim 14, wherein thebone fiber has a grain in the length direction of the fiber.
 27. Thebone material composition of claim 14, wherein the serrated edges andgrooves act as an effective binding substrate for the bone-formingcells.